Mycobacterium tuberculosis infection remains a major global health crisis. A multidisciplinary plan to control this epidemic must include new antibiotics and effective vaccination, but both of these therapeutic options are not presently available. Abundant recent evidence has implicated the M. tuberculosis cell envelope as an important pathogenesis determinant and therefore cell envelope biosynthetic pathways are an attractive therapeutic target. Data gathered during the prior award period of this grant defined the biosynthetic function and pathogenic importance of the mycolic acid methyltransferase enzyme family which modify the mycolic acids of M. tuberculosis with cyclopropane rings and methyl branches. Whereas loss of pcaA attenuates Mtb virulence, loss of cmaA2 and trans cyclopropanation causes hypervirulence and exacerbated granulomatous inflammation. While these results implicate cyclopropanation as an important pathogenesis determinant, they do not clearly recommend mycolic acid methyltransferase as a target for antibiotic development due to the unclear benefit to the host of loss of cyclopropanation. In this application we present preliminary data indicating that mycolic acid methyltransferases as an enzyme class are essential for slow growing mycobacterial viability in vitro. Genetic ablation of cmaA2 and mmaA3 in M. bovis BCG is synthetically lethal, suggesting a novel physiologic role for this lipid modification. Furthermore, we show that a chemical inhibitor of E. coli cyclopropane fatty acid synthase inhibits multiple pathways of mycolic acid cyclopropanation and inhibits mycobacterial growth at the same concentration. These results indicate that mycolic acid methyltransferases have a previously unrecognized essential function and that chemical inhibition of this enzyme class may be an attractive strategy for M. tuberculosis drug development. In this application we will build on these findings using genetics and biochemistry to substantiate mycolic acid methyltransferases as a target for antibiotic development and to understand the essential role of cyclopropanation in slow growing mycobacteria. PUBLIC HEALTH RELEVANCE: This project seeks to understand the function of a family of lipid modifying enzymes (mycolic acid cyclopropane synthases) in Mycobacterium tuberculosis, the causative bacterium of the disease Tuberculosis. Tuberculosis is a major global health problem and there is an urgent need for new antibiotics to treat this infection. The experiments in this application are designed to validate this enzyme family as a target for antibiotic development and to understand their role in causing disease.